<html> 1，血培养液标为“正” <ol><li>从连续监测温育箱中取出信号血培养瓶，并把它变成一个生物安全柜。注意：瓶可以含有有害微生物和普遍预防需要遵循。由于采样传染性气溶胶的危险性，所有抽样程序必须在生物安全II级层流柜中进行。 </li></ol> 2，革兰氏染色准备<ol><li>从信号血培养肉汤按当地机构的协议准备革兰氏染色。注意：当革兰氏阴性生物体的显微鉴定的血培养液处理为每个下面的方法。当革兰氏阳性菌鉴定，基因靶向识别和抗性标记另一种分子生物学方法应用于肉汤（本文中未提及）13。 </li></ol>标记的血培养肉汤电子“&gt; 3。转移到血清分离管<ol><li>轻轻颠倒2-3倍混合血培养瓶。 </li><li>在生物安全柜连接10 ml注射器的安全血液传输设备。 </li><li>附加的血液转移装置至血液培养瓶和撤回将5ml培养液进入注射器。转让吸气公元前清汤倒入血清分离管。 </li></ol>血培养液对4。浓度<ol><li>离心血清分离管中以1250×g离心15分钟，除去大量的红血细胞。 </li><li>吸出物，并用无菌移液管小心离开紧接凝胶/液体界面上方大约1毫升的血沉棕黄层的弃上清。注意：需氧瓶显示的红血细胞的明确分离到管的底部与凝胶/液界面出现白色不透明的颜色。相反，当加到凝胶/液体界面显示为深红色由于裂解红血细胞成分在上清液中余下的悬浮裂解厌氧血培养肉汤。 </li></ol> 5，重复离心洗涤步骤<ol><li>轻轻混合，最后加入1ml的血沉棕黄层的流体上面的凝胶界面，用无菌移液管，然后将整个体积转移到1.5ml的离心管中。离心新管中于288×g离心30秒。 </li><li>使用一次性塑料1毫升移液管插入一个新的1.5 ml微量离心管转移上清液并丢弃管与沉淀。注意：这是在该步骤中小心地避免粒料的转移是至关重要的。这是用于从厌氧公元前瓶子被处理的上清液仍颜料和颗粒并不总是清楚地看到试样尤为重要。 </li></ol>残余细胞6。裂解</p&gt; <ol><li>离心机的样品在18407×g离心1分钟，然后吸出物（和废弃），使用细尖移液管离开尽可能少的残余液体尽可能不破坏沉淀上清液。 </li><li>通过上下吹打重悬沉淀物残留在1ml无菌无DNA酶和RNA酶的游离水。注：有些作者所描述的使用酒精溶液代替无菌水重悬这使得细菌非可行的沉淀。 </li><li>离心悬浮液于18,407×g离心1分钟。 </li></ol>细菌蛋白质的7。提取<ol><li>吸弃上清液，用吸用细尖吸管确保尽可能多的液体尽量除去了。 </li><li>重悬沉淀于10μl甲酸（70％V / V）。注：甲酸可影响身体，如果它被吸入或者如果它与皮肤接触。在准备或操作的解决方案是为recommended员工使用通风柜除了个人防护装备。 </li><li>拌匀使用移液管，以确保均匀的悬浮液。使用移液管尖端的物理破坏的颗粒可能是必要的，以确保更均匀的溶液。 </li></ol> 8，准备MALDI-TOF的目标板<ol><li>接种干净的MALDI-TOF靶板与2μl每个目标点的准备暂停。制备3靶位点的每个样品，以克服失败读取偶然问题。 </li><li>让MALDI-TOF靶板干燥。干燥速率可以增大通过将板放在通风橱中的边缘，以最大化整个板的气流。 </li><li>覆盖各干燥点与1微升的基质溶液（10 mg / ml的α-氰基-4 - 羟基肉桂酸（HCCA），50％乙腈，2.5％三氟乙酸）。注：基质溶液可以影响身体，如果它被吸入或者如果它进入其所含克拉皮肤。在准备或操作的解决方案，建议员工使用通风柜除了个人防护装备。 </li><li>允许的MALDI-TOF靶板上干燥，如上所述的烟橱的边缘。确保均匀制剂填充每个平板上的目标点。 </li></ol> 9。地方MALDI-TOF靶板进入质谱仪<ol><li>将MALDI-TOF目标板进入MALDI-TOF质谱仪。确保板坐在弹簧加载板齐平。注意：请不要将目标板，直到所有的目标点都干。 </li><li>确保橡胶密封件是干净的线头，灰尘和毛发，使可以创建所需的真空条件。 </li><li>关闭MALDI-TOF阶段盖子，然后按下仪器前面的“输入/输出”按钮。盖子现在将锁定并允许创建必要的真空度。 </li></ol> 10，MALDI-TOF MS Spectra收购<ol><li>打开MALDI生物分型和FlexControl软件。 </li><li>内的打字软件请从下拉菜单中选择“新分类”标题为“文件”。将项目命名，然后选择“新建”。选择“下一步”的打字软件内完成设置。 </li><li>在控制软件窗口识别屏幕上的图形目标板。突出显示MALDI-TOF目标点正在使用通过移动鼠标在接种点，同时按住鼠标左键。 </li><li>用鼠标右键单击任意位置上选定的目标位置，然后选择从下拉菜单中选择“添加分析物”。 </li><li>加血培养鉴定的详细信息的“ID”列中为每个目标点，然后点击“下一步”完成时。 </li><li>选择商业“分类学”光谱数据库，然后选择“下一步”。注：Labo的ratories可以利用额外的内部或商业数据库，以提高参考光谱的数目。 </li><li>点击“完成”和MALDI-TOF MS将开始产生光谱。注意：MALDI-TOF设置为按制造商的设定（线性正模式，60赫兹激光频率，20千伏的加速电压，16.7千伏电压IS2，170毫微秒的提取延迟和2,000-20,137 m / z范围）。 </li><li>如果MALDI控制软件无法生成的山峰，人工采集的光谱可以进行（没有描述的方法）。 </li></ol> 11发布分析MALDI-TOF MS分数的解释<ol><li>一旦光谱采集和分析完成后，请在打字的软件旁边的物种鉴定的“+”按钮，展开对每个目标的识别列表。 </li><li>研究了前5名标识，并指出，如果前5名是相似的。不和谐的前5个属，分数≥1.7相同的目标点引发混合肉汤的可能性。注：MALDI-TOF技术具有敏感性差检测混合肉汤。例如，当发酵液包含混合物种高置信度得分可以被识别为混合种中的一种。 </li><li>当得分≥2.0是注意到在目标点的最高匹配，报告该物种。 </li><li>当最高得分上的目标点为≥1.7，但&lt;2.0，只报告了属。如果前5名标识的附加条件是一致的，然后向种的水平。 </li></ol> 12，去除MALDI-TOF靶板<ol><li>当所有的光谱是完全按MALDI-TOF机器上的“IN / OUT”按钮。 </li><li>打开MALDI-TOF靶板插座和移除MALDI-TOF靶板。 </li><li>如果使用的是可重复使用的MALDI-TOF靶板，用酒精清洁所有目标点和MALDI-TOF板储存在室温，一旦它的清洁和干燥。 </li></ol>

<ol>
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作者宣称，他们有没有竞争的财务权益。

运用MALDI-TOF MS到后离心步骤都与足够的照顾不进行重新混合分离的成分血培养肉汤时，这一点很重要。特别重要的是，除去血培养成分和人类细胞的蛋白质，包括血红蛋白，这可能会产生尖峰与MALDI-TOF光谱干扰。 虽然MS制造商推荐切得分≥2.0的物种和≥1.7，属鉴定，其他报告表明较低的对数得分（范围≥1.4至≥1.6）时，适用于公元前肉汤10,12,14-18可以实现。在临床微?...

由于细菌患者的血流感染（BSI）继续有较高的住院死亡率，范围从6-48％1。适当的经验性抗生素交付促进生存和在严重脓毒症患者的子集，每个小时的延迟，以适当的治疗相关的生存2,3下降。因此，临床实验室的主要目标是快速检测，识别和沟通细菌在血培养存在的，告知临床决策。它已经证明，微生物实验室对抗菌治疗在报告的革兰氏染色4，最近的一次影响最大，观察研究证明，飞行质谱基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）直接在血培养液进行影响超过三分之一BSI引起的革兰阴性菌5订明。 ，6,7微生物的鉴定。该技术是目前公认，并已集成到许多实验室进行快速准确的识别微生物中分离固体培养基上6,8。直接应用MALDI-TOF MS对血培养（BC）的肉汤已经表示“积极”的微生物，因为获得低成本的早期识别微生物的潜在的临床医生和实验室管理人员的上诉。 直接应用MALDI-TOF MS的血培养液的临床应用已被广泛观察到的灵敏度，当与鉴定标准表型文化为基础的方法，以成功识别革兰阴性菌不等的报告相比，限制从47-98.9 ％9-11。在灵敏度的变化可能涉及对BC肉汤组成，初始细菌浓度，变化的样品制备方法，以及在研究人群9中遇到的革兰氏阴性生物体的数组。与这些其他的发布的协议相比，这里提出的方法可避免使用乙醇，氯化铵或附加的（非矩阵）乙腈。其结果是将细菌沉淀将保持存活（直到蛋白提取的点），允许潜在敏感性表型试验方法，可直接应用于这些微生物在液体培养基。此外，所提出的方法已经被证明是便宜的，可靠的，快速，细菌鉴定内的血培养物革兰氏染色结果25分钟内可用的，以最小的手上的时间12。 这种方法是利用甲酸提取一个简单的内部自旋裂解方案直接应用到血培养阳性肉汤识别革兰氏阴性bacteria与MALDI-TOF MS技术。

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

临床微生物学实验室的一个重要作用是提供快速鉴定细菌，导致血液感染的。传统的识别需要信号血培养肉汤识别可用的亚文化不仅已经成熟后，在固体琼脂菌落。 MALDI-TOF MS是用于识别大多数临床相关的细菌时，适用于菌落在固体培养基上可靠的，快速的方法。 MALDI-TOF MS直接到血培养液中的应用是一个有吸引力的方法，因为它有潜力加速了细菌的种属鉴定，提高临床管理。然而，要克服的一个重要问题是，在分析前除去包含在血培养标本，如不去除，干扰MS谱，并可能导致判别识别分数不足或低干扰的树脂，蛋白质和血红蛋白。此外，有必要集中硝化细菌IA开发足够的质量谱。该方法描述了浓缩，纯化和提取的革兰氏阴性细菌，允许从一个信号血液培养液中的早期识别细菌。

所生成的MALDI-TOF MS谱进行比较，以光谱的集成参考数据库。对数分数分配给测试分离和参考数据库隔离之间的比赛的信心，用分数≥1.7所需的鉴定可能要属水平（ 图1），≥2.0为可能的识别物种（ 图的建议2）。报告以种的水平时，得分≥1.7和所述第一5标识所匹配的打字软件是一致的（ 图1）12。图3演示了当混合物种的血培养液进行分析?...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

的飞行（MALDI-TOF）质谱（MS），直接向血培养肉汤基质辅助激光解吸/电离飞行时间质谱的应用加快细菌的鉴定。该方法是一种快速和可靠的方法，可直接从血培养肉汤鉴定革兰氏阴性细菌。

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

rapid-identification-gram-negative-bacteria-from-blood-culture-broth

Research

JoVE Journal

Immunology and Infection

23.8K 次观看.  Westmead Hospital. 的飞行（MALDI-TOF）质谱（MS），直接向血培养肉汤基质辅助激光解吸/电离飞行时间质谱的应用加快细菌的鉴定。该方法是一种快速和可靠的方法，可直接从血培养肉汤鉴定革兰氏阴性细菌。 

视频: Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

一个1.5小时的程序，鉴定肠球菌直接从血培养的物种

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

科研

免疫与感染

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

全细胞MALDI-TOF质谱法是一种准确，快速的方法来分析巨噬细胞活化的不同模式

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

从肾脏双重否定αβ性T细胞的分离

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

为快速细菌鉴定和药敏试验的准备血培养微丸

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

参考微量稀释法达巴万星<em&gt;体外</em&gt;细菌敏感性测试生长有氧

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...